Tissue physiology and the response to heat

Abstract

The most important physiological parameter influencing tissue response to heat is blood flow. At mild hyperthermia temperatures blood perfusion increases in many tumours and this effect is heating time-, temperature- and tumour-dependent. These flow increases can improve tumour oxygenation.

 When heating is terminated, perfusion and oxygenation commonly recover, although how quickly this occurs appears to be tumour-specific. While these effects are unlikely to have any anti-tumour activity they can be exploited to improve the combination of heat with other therapies. However, since similar physiological effects should occur in normal tissues, such combination therapies must be carefully applied. Heating tumours to higher temperatures typically causes a transient increase in perfusion during heating, followed by vascular collapse which if sufficient will increase tumour necrosis. The speed and degree of vascular collapse is dependent on heating time, temperature and tumour model used.

 Such vascular collapse generally occurs at temperatures that cause a substantial blood flow increase in certain normal tissues, thus preferential anti-tumour effects can be achieved. The tumour vascular supply can also be exploited to improve the response to heat. Decreasing blood flow, using transient physiological modifiers or longer acting vascular disrupting agents prior to the initiation of heating, can both increase the accumulation of physical heat in the tumour, as well as increase heat sensitivity by changing the tumour micro-environmental parameters, primarily an increase in tumour acidity.

 Such changes are generally not seen in normal tissues, thus resulting in a therapeutic benefit.

• Hyperthermia
• physiological effects
• blood flow
• physiological modifiers
• vascular disrupting agents

Introduction

There is a strong relationship between tissue physiology and the response of that tissue to heating. The most important physiological parameter in this context is blood flow. When any tissue is heated various physiological changes occur, the majority of which are secondary to changes in blood flow [1]. Blood flow is also one of the major vehicles by which heat is dissipated from tissues, thus the tissue blood supply will have a significant influence on the ability to heat that tissue [2]. In general, the lower the rate of blood flow then the easier it is to heat. Blood flow is also important in determining the micro-environmental conditions found within tissues, especially in tumours [3], [4]. Although solid tumours can have blood flow values that can be greater than that of certain normal tissues, when compared to normal tissues the tumour blood supply is generally primitive and chaotic in nature, which can result in areas that are nutrient-deprived, low in oxygen and highly acidic and cells that exist in these adverse conditions are generally more sensitive to the cytotoxic effect of heat. The aim of this presentation will be to briefly review the physiological changes that occur in following heating and how the physiology can be exploited to improve the response to hyperthermia.

Physiological consequences of heating

The physiological changes that occur when tumours are heated at different temperatures is illustrated in Figure 1. Mild heat temperatures cause an increase in blood flow. At the same time there is a corresponding increase in the oxyhaemoglobin saturation of the individual red blood cells within tumour micro-vessels. These changes will lead to an overall increase in oxygen availability. Other studies have also shown that mild hyperthermia temperatures improve tumour oxygenation status [5], [6]. At higher temperatures one may see a very transient increase in blood flow during the heating period, but vascular damage soon begins to occur and this will rapidly lead to a decrease in tumour blood flow [2], [7], [8]. With higher heat temperatures there is also a corresponding decrease in oxyhaemoglobin saturation, and these changes will result in a decrease in overall oxygen availability. This lack of oxygen will also give rise to a decrease in tumour pH and ultimately lead to ischemia and cell death [3]. The degree of vascular shut-down at the higher temperatures appears to be dependent on the time and temperature of heating. It is also dependent on the tumour type, and this may be a reflection of the tumour blood flow prior to treatment, since it has been shown that the lower the pre-treatment flow then the more severe the heat induced shut-down [9]. Normal tissues typically show a very different vascular response to heat, with flow essentially increasing as the temperature increases, even at temperatures that produce substantial vascular occlusion in tumours [7], [9].

Figure 1. Physiological changes induced during heating of tissue-isolated tumour preparations as a function of tumour temperature. Results show tumour blood flow (TBF), haemoglobin-oxygen (HbO₂) saturation in tumour micro-vessels, tumour oxygen availability and mean intra-tumour oxygen partial pressure (pO₂). (Adapted from [1].)

Probably the most controversial aspect of the physiological effects of hyperthermia is how long the improvements in tumour oxygen at mild hyperthermia treatments actually last. In a C3H mouse mammary carcinoma, tumour oxygenation returned to normal within a matter of minutes after the heating was stopped [6]. Others have shown that the enhanced oxygenation can actually last for up to 24-h after heating [5], although this effect is difficult to explain because the physiological changes that account for the enhanced tumour oxygenation at these temperatures are unlikely to be prolonged. There is even clinical data that seems to support the argument for a prolonged improvement in oxygenation at mild temperatures [10], [11]. However, those clinical studies also showed that this increased oxygenation correlated with the degree of damage seen, and an additional pre-clinical study clearly demonstrated enhanced oxygenation 24–48 h after heating at temperatures and treatment times that were severe and caused substantial tumour control, presumably due to vascular shut-down that would have been expected with such treatments [12]. This may suggest that the apparent improvements in oxygenation in the clinical studies may simply be reflecting damage resulting from the higher temperatures and not ‘reoxygenation’ at lower temperatures.

Exploiting tumour physiology to improve heating

Since blood flow to tissues influences both the ability to heat the tissue and the damage induced by heat, it naturally follows that modifying blood flow should be a biologically valid approach for improving hyperthermia. Numerous studies have now shown that decreasing tumour blood flow either by physical clamping [13–15] or physiological modification using high-dose hydralazine [16–20], sodium nitroprusside [21–23] or glucose [24–27] are effective at improving tumour response to hyperthermia. This is illustrated in Figure 2 for hydralazine (HDZ), in which heating tumours at the time of maximal vascular shut down by HDZ increases the slope of the heat dose-response curve and that this occurs at all temperatures from 40.5–43.5°C. Similar effects are unlikely to occur in normal tissues, primarily because such physiological modifiers do not induce significant reductions in normal tissue blood flow [30]. However, despite the therapeutic benefit of these approaches, the clinical applicability of such methods remains questionable.

Figure 2. The effect of blood flow modifiers on the response of a C3H mammary carcinoma to heat. Tumours were locally heated at 42.5°C + drugs for up to 180 min and the tumour growth time (time taken for tumours to grow from 200–1000 mm³) calculated (left panel), with the results shown as means (+1 SE) for an average of 12 animals per group. The slope values for these curves and for similar data at other temperatures were then calculated and are shown (right panel). For both figures the results are for heat alone (○) or heating started 30 min after injecting HDZ (▴; 5 mg kg⁻¹; i.v.) or 3 h after FAA (•; 150 mg kg⁻¹; i.p.). (Adapted from [17], [28], [29] and unpublished data.)

A more clinically relevant approach may be the use of vascular disrupting agents (VDAs). These damage tumour vasculature and thus reduce blood flow, without producing similar effects in normal tissues [28], [31–35], and several VDAs are currently in clinical testing [36]. Many of these VDAs have also been combined with hyperthermia, including tumour necrosis factor [37], [38], arsenic trioxide [39], [40], vinblastine [41], flavone acetic acid [28], [29], 5,6-dimethylxanthenone-4-acetic acid [42] and combretastatin A-4 disodium phosphate [43–45] and in every case there was an enhancement of the heat response. This effect was time- and schedule-dependent, with the maximum response generally observed if the heating was started 1–6 h after VDA injection [28], [29], [39], [40], [42], [42–46] and this clearly corresponded to the time of maximal reduction in tumour blood flow in those studies. Injecting VDAs after heating has been shown to be substantially less effective at enhancing the heat response [28], [42], [45], [46] and in most cases did not appear to be greater than a simple additive effect of the VDA and heat alone. Typical results illustrating the enhancement of heat damage by VDAs are shown in Figure 2 using flavone acetic acid (FAA) as the example. As with most VDAs, FAA alone had a small effect on tumour growth, but when tumours were heated at the time of maximal vascular shut down there was a significant increase in the heat dose-response curve. Similar to HDZ, the FAA induced enhancement of heat damage is temperature dependent (Figure 2). However, although the enhancement by FAA at higher temperatures resembled the effect seen with HDZ, at mild hyperthermia treatments the effect is much larger. Similar enhancements have been seen with other VDAs [42], [45]. Why this should be is not entirely clear. The reductions in tumour blood flow induced by HDZ and VDAs can lead to a better tumour heating [17], [19], [20], [41], [46]. However, as a result of the vascular shut down there is a corresponding decrease in tumour pH with both HDZ and VDAs [[46], [47], Horsman and Maxwell unpublished observations] and since it is well known that cells under low pH are more sensitive to killing by heat [48], [49], part of the enhanced heat response is related to an increase in tumour damage [17], [18], [20], [28], [29], [42], [45] and this latter effect may play the more significant role. However, some studies revealed that heat damage could be maximally enhanced in situations where the effects on tumour perfusion were well below maximal [41], [45]. This suggests that unlike the situation with physiological modifiers of tumour blood flow, the enhancement of heat damage by VDAs involves other, as yet unknown, factors unrelated to a simple reduction in tumour blood flow, and it may be these factors which account for the differences at mild temperatures.

Some studies have investigated the possible enhancement of heat damage by VDAs in normal tissues. In general, VDAs do enhance heat response in such tissues [28], [42–45]. However, these increases have always been far less than those seen in tumours, thus resulting in a therapeutic benefit.

Conclusions and clinical relevance

Hyperthermia on its own had no role to play in the curative treatment of tumours in humans and its clinical potential lies in its use as an adjuvant with other more conventional treatments [50]. Indeed, several clinical studies have demonstrated the benefit of combining hyperthermia with radiation or chemotherapy [51–53]. However, the physiological changes induced by heating could be better exploited to improve this. High temperatures decrease blood flow and could be used to trap chemotherapeutic drugs in tumours. There is also a decrease in tumour pH and this is known to increase the sensitivity of certain drugs [54]. Mild hyperthermia treatments increase tumour perfusion and this could be utilised to improve drug delivery. At the same time the increase in tumour oxygenation would be expected to increase radiation sensitivity; however, this would probably require giving the heat at the same time or immediately prior to irradiating, which is not how current schedules are used.

With the blood flow modifiers, especially the VDAs, the greatest advantage lies in the fact that, when combined with mild heat treatments of ∼41.5°C, they produce effects that are similar to those seen at temperatures of 42.5–43°C alone. Clinically, temperatures of ∼43°C are generally considered necessary for efficacy [52], but it is often difficult to achieve this temperature and less effective mild temperatures of ∼41.5°C are often seen [55]. Vascular modifiers thus appear to be an effective method for converting the mild temperatures into a more efficient therapy, which suggests that if future clinical trials with hyperthermia were to include such agents in the treatment regime, we may overcome the problem of ineffective heating of tumours and many more trials should be beneficial.

Acknowledgements

This presentation was made possible by financial support from the Danish Cancer Society and the Danish Research Agency.